Proteomics Multidimensional LC Solutions

Using the unique x2 dual-gradient technology, 2D-LC methods can be performed on a single system. Use the switching valves inside the NCS-3500 column compartment for on-line 2D-LC or combine the x2 dual-gradient technology with the micro fractionation option to perform off-line 2D-LC. The UltiMate® 3000 Proteomics MDLC offers a wide flow rate range and high flow rate flexibility, allowing the system to perform an extensive range of applications, as shown in the figure below:

2D-LC Salt Steps

The 2D-LC salt step solution is the easy-to-use, flexible solution for moderately complex proteomics analyses. It allows 2-D separations by using the autosampler and salt injections to transfer fractions from the first to the second dimension.

The 2D-LC salt step LCi solution is based on injections of salt plugs onto an ion-exchange column. Typically, a protein digest is injected onto a strong cation-exchange column for separation in the first dimension. Peptides are eluted and separated as fractions by injecting salt plugs with increasing salt concentrations. Each fraction is subsequently separated on a reversed-phase column in the second, orthogonal dimension. The total number of salt plugs used depends on the sample complexity. This method can be fully automated. 2D-LC salt step kits are available for nano, capillary, and monolithic configurations. The same fluidic configuration also supports Proteomics Preconcentration.

Flow schematic for 2D-LC salt steps.

2D-LC Off-Line

The off-line 2D-LC solution supports the separation of highly complex proteomic samples. The advantages of off-line MDLC techniques over on-line approaches include high flexibility in column dimensions and mobile-phase compositions, as well as the ability to re-analyze sample fractions. The micro fraction collection option of the WPS-3000PL Nano/Cap Pulled Loop autosampler supports fully automated off-line 2D-LC, a unique solution for the analysis of complex proteomics samples.

The typical workflow for automated off-line MDLC involves sample injection and separation in the first dimension, collection of eluting peaks by the WPS autosampler, re-injection, and subsequent separation in the second dimension. The analytes are finally detected by UV and MS detection. Off-Line 2D-LC kits are available for peptides (SCX-RP) and proteins (WAX-RP).